Dilated Cardiomyopathy Mutations Research Articles

Integrated machine learning and multimodal data fusion for patho-phenotypic feature recognition in iPSC models of dilated cardiomyopathy.

Integration of multiple data sources presents a challenge for accurate prediction of molecular patho-phenotypic features in automated analysis of data from human model systems. Here, we applied a machine learning-based data integration to distinguish patho-phenotypic features at the subcellular level for dilated cardiomyopathy (DCM). We employed a human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model of a DCM mutation in the sarcomere protein troponin T (TnT), TnT-R141W, compared to isogenic healthy (WT) control iPSC-CMs. We established a multimodal data fusion (MDF)-based analysis to integrate source datasets for Ca2+ transients, force measurements, and contractility recordings. Data were acquired for three additional layer types, single cells, cell monolayers, and 3D spheroid iPSC-CM models. For data analysis, numerical conversion as well as fusion of data from Ca2+ transients, force measurements, and contractility recordings, a non-negative blind deconvolution (NNBD)-based method was applied. Using an XGBoost algorithm, we found a high prediction accuracy for fused single cell, monolayer, and 3D spheroid iPSC-CM models (≥92±0.08 %), as well as for fused Ca2+ transient, beating force, and contractility models (>96±0.04 %). Integrating MDF and XGBoost provides a highly effective analysis tool for prediction of patho-phenotypic features in complex human disease models such as DCM iPSC-CMs.

Abstract 16649: Clonal Hematopoiesis as a Predictor of Left Ventricular Reverse Remodeling in Non-Ischemic Dilated Cardiomyopathy

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is an independent risk factor of major adverse cardiovascular events, including heart failure (HF) with reduced left ventricular ejection fraction (LVEF). Although genetic mutations are known to affect clinical prognosis and left ventricular reverse remodeling (LVRR) in non-ischemic dilated cardiomyopathy (DCM), the prevalence and clinical significance of CHIP in DCM remain elusive. Objectives: The principal aim of this study is to delineate the distribution and prognostic impact of CHIP in non-ischemic DCM. Methods: We included 198 cases of DCM with LVEF ≤ 40% and no identifiable cause for left ventricular systolic dysfunction from the Japanese multi-center cohort. Whole exome sequencing was performed for germline analysis, and deep target sequencing focusing on CHIP driver genes for somatic analysis was conducted. We detected cardiomyopathy-related pathogenic mutations and CHIP driver mutations using GATK best practices and Mutect2, respectively. The primary endpoint was LVRR, defined as an absolute increase in LVEF by ≥ 10% at a one-year follow-up echocardiogram. Results: Twenty-seven CHIP driver mutations were detected in 24 (12%) of the DCM patients. The mean read coverage for CHIP driver mutation was 2156. DNMT3A was the most frequent mutated gene in CHIP, followed by TET2 . Ninety-two (46%) patients have cardiomyopathy-related pathogenic mutations. TTN was the most frequent mutated gene, followed by LMNA . Although the CHIP-positive group showed similar LVEF and prevalence of cardiomyopathy-related pathogenic mutations compared with the CHIP-negative group, the occurrence of LVRR was significantly lower in the CHIP-positive group (47.0% vs. 20.8%, P=0.018). Multivariate logistic regression analysis revealed that CHIP (odds ratio (OR): 0.23, P=0.022) and cardiomyopathy-related pathogenic mutations (OR: 0.28, P<0.001) were independent negative predictors of LVRR. Conclusions: Determining germline and somatic mutations in DCM significantly predict clinical prognosis.

An Alternative Mechanism of Subcellular Iron Uptake Deficiency in Cardiomyocytes.

Systemic defects in intestinal iron absorption, circulation, and retention cause iron deficiency in 50% of patients with heart failure. Defective subcellular iron uptake mechanisms that are independent of systemic absorption are incompletely understood. The main intracellular route for iron uptake in cardiomyocytes is clathrin-mediated endocytosis. We investigated subcellular iron uptake mechanisms in patient-derived and CRISPR/Cas-edited induced pluripotent stem cell-derived cardiomyocytes as well as patient-derived heart tissue. We used an integrated platform of DIA-MA (mass spectrometry data-independent acquisition)-based proteomics and signaling pathway interrogation. We employed a genetic induced pluripotent stem cell model of 2 inherited mutations (TnT [troponin T]-R141W and TPM1 [tropomyosin 1]-L185F) that lead to dilated cardiomyopathy (DCM), a frequent cause of heart failure, to study the underlying molecular dysfunctions of DCM mutations. We identified a druggable molecular pathomechanism of impaired subcellular iron deficiency that is independent of systemic iron metabolism. Clathrin-mediated endocytosis defects as well as impaired endosome distribution and cargo transfer were identified as a basis for subcellular iron deficiency in DCM-induced pluripotent stem cell-derived cardiomyocytes. The clathrin-mediated endocytosis defects were also confirmed in the hearts of patients with DCM with end-stage heart failure. Correction of the TPM1-L185F mutation in DCM patient-derived induced pluripotent stem cells, treatment with a peptide, Rho activator II, or iron supplementation rescued the molecular disease pathway and recovered contractility. Phenocopying the effects of the TPM1-L185F mutation into WT induced pluripotent stem cell-derived cardiomyocytes could be ameliorated by iron supplementation. Our findings suggest that impaired endocytosis and cargo transport resulting in subcellular iron deficiency could be a relevant pathomechanism for patients with DCM carrying inherited mutations. Insight into this molecular mechanism may contribute to the development of treatment strategies and risk management in heart failure.

Investigating mutant hydrophobic patch exposure in troponin C using molecular dynamics-based free energy methods.

Contraction of cardiomyocytes is orchestrated through calcium-binding to the N-terminal regulatory domain of cardiac troponin C (NcTnC). This binding event initiates a series of dynamic and conformational changes that expose a hydrophobic patch to which troponin I (TnI) associates. Cardiomyopathies have been linked to mutations in NcTnC that disrupt these precise interactions and modulate free energy of opening. Mutations that lower the free energy of opening have a calcium-sensitizing effect and those that increase free energy of opening have a calcium-desensitizing effect, like mutations implicated with hypertrophic (HCM) and dilated cardiomyopathy (DCM), respectively. Albeit mutations occurring in the loop region of NcTnC have been most extensively studied, information regarding mutations within the hydrophobic patch is lacking. Simulating the closed-to-open transition in NcTnC is challenging and typically requires microsecond timescales just to observe partial opening of the patch. Here, we use targeted molecular dynamics (TMD) to efficiently sample the landscape between the open and closed conformations. Umbrella sampling (US) was employed to predict the free energy of opening by using frames sourced from TMD, and preliminary data corroborates the expected trends for known HCM and DCM mutations in NcTnC. RosettaDesign was used to prepare and score novel mutation candidates by systematically mutating residues at eight different positions in the patch to almost every other amino acid. Based on the ref15_cart scoring function, up to three mutation candidates from each position were selected to proceed to further evaluation with US. In this work, we identify several mutations that are expected to perturb patch opening and present an innovative approach to quantitatively study the influence hydrophobic patch residues play in the molecular basis of myocyte contraction.

Dilated cardiomyopathy mutation (E525K) in human beta-cardiac myosin enhances actin-activated phosphate release but stabilizes the auto-inhibited super-relaxed state.

Mutations in beta-cardiac myosin (M2β) are a common cause of inherited cardiomyopathies including dilated (DCM) and hypertrophic cardiomyopathy (HCM). We investigated the impact of a DCM mutation (E525K) in the human M2β motor domain, which has been shown to stabilize both the interacting heads motif (IHM) and auto-inhibited super-relaxed (SRX) state via head-tail electrostatic interactions in 15 heptad heavy meromyosin (M2β HMM). These findings suggest E525K may reduce muscle force and power by triggering an increase in the IHM/SRX state. However, it is unclear how E525K impacts the intrinsic motor properties of M2β. Thus, we introduced the mutation into single-headed human cardiac myosin subfragment 1 (M2β-S1), unable to form the IHM, and examined its impact on the ATPase mechanism. We revealed that E525K induces a 2.8-fold increase in maximum steady-state actin-activated ATPase activity (kcat), a 6-fold decrease in the actin concentration at which ATPase is half maximal (KATPase), and a 3.4-fold increase in the fast phase of actin-activated phosphate release. Glutamate 525 is located in the conserved activation loop that is proposed to trigger phosphate release by interacting with the negatively charged N-terminus of actin. We propose the mutation may enhance the rate of rotation of the lower 50-kDa domain, movement of the relay helix/switch II, and lever arm rotation associated with the power stroke. Thus, we will directly examine the impact on the actin-activated power stroke utilizing a FRET approach that measures lever arm rotation. Our results suggest the E525K mutation triggers an increase in intrinsic motor properties, while its ability to stabilize the SRX/IHM state in the myosin dimer likely dominates the molecular mechanism that leads to decreased muscle force and power and DCM pathogenesis.

Cryo-EM structure of tarantula myosin filament: Implications for analysis of myopathy mutations.

The high-resolution structure of myosin thick filaments of invertebrates has aided understanding of key muscle functions and analysis of disease mutations in humans. We previously published a low-resolution tarantula skeletal thick filament cryo-EM structure (Woodhead et al., 2005), which was used to map hypertrophic and dilated cardiomyopathy mutations (Alamo et al., 2017, Nag et al., 2017), present mostly in the head-head and head-tail interaction sites of the interacting-heads motif (IHM). This study suggested that the myosin coiled-coil tails in the filament backbone were arranged in groups of three, forming 40-Å-diameter subfilaments (Wray, 1979), but it lacked the detail required to resolve individual tails and to map the 40% of myopathy mutations that occur in the tail region of the molecule. Here we report a 5.5 Å resolution cryo-EM map of the tarantula thick filament backbone that resolves the densities of the full length of the myosin tails and their packing interactions. The map reveals that the tails do not form subfilaments but are arranged individually in layers forming a “molecular crystal,” in which the myosin coiled-coils gradually move towards the center of the filament, near the paramyosin core, while slowly curving around the filament axis. A similar arrangement was found in insect flight muscle thick filaments (Hu et al., 2016), suggesting it could be a general packing scheme in thick filaments, as originally proposed by Squire (1973). The positions of skip residues predict longer pitch regions and bending of the coiled-coils, as seen in the cryo-EM density. The structure also suggests new interactions of the blocked-head converter region with the backbone of the filament, implying hyperstability of IHMs. Improved resolution, and solution of the vertebrate thick filament structure, should enable precise mapping of tail mutations.

A dilated cardiomyopathy mutation of phospholamban, R14del, increases phospholamban pentamer stability.

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca2+ stores in cardiac muscle. Phospholamban (PLB), a micropeptide inhibitor of SERCA, forms avid homo-pentamers, but it is the monomeric form that regulates the pump. The dynamic exchange of monomeric PLB between pentamers and the regulatory complex with SERCA is an important determinant of cardiac responsiveness to exercise. Here, we investigated two naturally occurring pathogenic mutations of PLB, a cysteine substitution of arginine 9 (R9C) and an in-frame deletion of arginine 14 (R14del). Both of these mutations are associated with dilated cardiomyopathy. We previously showed that the R9C mutation causes disulfide crosslinking and hyperstabilization of pentamers. While the pathogenic mechanism of R14del is unclear, we hypothesized that this mutation may also alter PLB homo-oligomerization and disrupt the PLB-SERCA regulatory interaction. SDS-PAGE revealed a significantly increased pentamer:monomer ratio for R14del-PLB (87.2±3.2% pentamers) when compared to wild-type PLB (79.5±0.8% pentamers). In addition, we quantified homo-oligomerization and SERCA-binding in live cells using fluorescence resonance energy transfer (FRET) microscopy. R14del-PLB showed an increased affinity for homo-oligomerization and decreased binding affinity for SERCA compared to WT, suggesting that, like R9C, the R14del mutation traps PLB in its pentameric form, decreasing its ability to regulate SERCA. Moreover, the R14del mutation reduces the rate of PLB unbinding from the pentamer after a transient Ca2+ elevation, limiting the rate of re-binding to SERCA. A computational model predicted that hyperstabilization of R14del-PLB pentamers impairs the ability of cardiac Ca2+ handling to respond to changing heart rates between rest and exercise. We theorize that reduced responsiveness may contribute to arrhythmogenesis in human carriers of the R14del mutation.

Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting-heads motif and super-relaxed state of myosin.

The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain electron microscopy and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin.

DCM and HCM mutations to TPM1 differentially alter contractile behavior and gene expression profiles in engineered heart tissues

Hypertrophic (HCM) or dilated cardiomyopathy (DCM) can be caused by missense mutations in alpha-tropomyosin (TPM1). In this study, we focus on the TPM1 mutations E62Q (HCM) and E54K (DCM), which were introduced homozygously into separate induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9. iPSC-derived cardiomyocytes were seeded into decellularized myocardial sections to form human engineered heart tissues (EHT) that are suitable for biomechanical evaluation of muscle phenotypes. After two weeks in culture, E62Q EHTs displayed a nearly 3-fold increase in peak isometric twitch force relative to isogenic control with no change in relative slope of length dependent activation.

Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting heads motif and super-relaxed state of myosin

Formation of the auto-inhibited super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We found that a single point mutation, E525K, associated with dilated cardiomyopathy (DCM) dramatically enhances the stability of the SRX state. We expressed human cardiac myosin containing the subfragment 1 domain, 15 heptads of the subfragment 2 coiled-coil region and a leucine zipper that facilitates dimerization, as well as a C-terminal GFP tag (M2β 15HPZ.GFP).

Harnessing multiscale models of a dilated cardiomyopathy mutation for precision medicine

Familial dilated cardiomyopathy (DCM) is a leading cause of heart failure in both adult and pediatric patients. Currently, there is no cure for DCM, and the 5-year transplant free survival rate is <50%. Therefore, there is an outstanding need to develop new therapeutics that improve outcomes in patients. Previous studies have identified causative mutations in a diverse set of cardiac genes that includes sarcomeric, mechanosensing, structural, and metabolic genes. The disease presentation can vary between individual genotypes.

Abstract P459: Mavacamten And Danicamtiv Reverse Respective Contractile Abnormalities In Engineered Heart Tissue Models Of Hypertrophic And Dilated Cardiomyopathy

Missense mutations in alpha-tropomyosin (TPM1) can lead to development of hypertrophic (HCM) or dilated cardiomyopathy (DCM). HCM mutation E62Q and DCM mutation E54K have previously been studied extensively in experimental systems ranging from in vitro biochemical assays to animal models, although some conflicting results have been found. We undertook a detailed multi-scale assessment of these mutants that included atomistic simulations, regulated in vitro motility (IVM) assays, and finally physiologically relevant human engineered heart tissues. In IVM assays, E62Q previously has shown increased Calcium sensitivity. New molecular dynamics data shows mutation-induced changes to tropomyosin dynamics and interactions with actin and troponin. Human engineered heart tissues (EHT) were generated by seeding iPSC-derived cardiomyocytes engineered using CRISPR/CAS9 to express either E62Q or E54K cardiomyopathy mutations. After two weeks in culture, E62Q EHTs showed a drastically hypercontractile twitch force and significantly increased stiffness while displaying little difference in twitch kinetics compared to wild-type isogenic control EHTs. On the other hand, E54K EHTs displayed hypocontractile isometric twitch force with faster kinetics, impaired length-dependent activation and lowered stiffness. Given these contractile abnormalities, we hypothesized that small molecule myosin modulators to appropriately activate or inhibit myosin activity would restore E54K or E62Q EHTs to normal behavior. Accordingly, E62Q EHTs were treated with 0.5μM mavacamten (to remedy hypercontractility) and E54K EHTs with 0.5 μM danicamtiv (to remedy hypocontractility) for 4 days, followed by a 1 day washout period. Upon contractility testing, it was observed that the drugs were able to reverse contractile phenotypes observed in mutant EHTs and restore contractile properties to levels resembling those of the untreated wild type group. The computational, IVM and EHT studies provide clear evidence in support of the hyper- vs. hypo-contractility paradigm as a common axis that distinguishes HCM and DCM TPM1 mutations. Myosin modulators that directly compensate for underlying myofilament aberrations show promising efficacy in human in vitro systems.

Myosin dilated cardiomyopathy mutation S532P disrupts actomyosin interactions, leading to altered muscle kinetics, reduced locomotion, and cardiac dilation in Drosophila.

Dilated cardiomyopathy (DCM), a life-threatening disease characterized by pathological heart enlargement, can be caused by myosin mutations that reduce contractile function. To better define the mechanistic basis of this disease, we employed the powerful genetic and integrative approaches available in Drosophila melanogaster. To this end, we generated and analyzed the first fly model of human myosin–induced DCM. The model reproduces the S532P human β-cardiac myosin heavy chain DCM mutation, which is located within an actin-binding region of the motor domain. In concordance with the mutation’s location at the actomyosin interface, steady-state ATPase and muscle mechanics experiments revealed that the S532P mutation reduces the rates of actin-dependent ATPase activity and actin binding and increases the rate of actin detachment. The depressed function of this myosin form reduces the number of cross-bridges during active wing beating, the power output of indirect flight muscles, and flight ability. Further, S532P mutant hearts exhibit cardiac dilation that is mutant gene dose–dependent. Our study shows that Drosophila can faithfully model various aspects of human DCM phenotypes and suggests that impaired actomyosin interactions in S532P myosin induce contractile deficits that trigger the disease.

Dilated Cardiomyopathy Mutations and Phosphorylation disrupt the Active Orientation of Cardiac Troponin C

Cardiac troponin (cTn) is made up of three subunits, cTnC, cTnI, and cTnT. The regulatory N-terminal domain of cTnC (cNTnC) controls cardiac muscle contraction in a calcium-dependent manner. We show that calcium-saturated cNTnC can adopt two different orientations, with the “active” orientation consistent with the 2020 cryo-EM structure of the activated cardiac thin filament by Yamada et al. Using solution NMR 15N R2 relaxation analysis, we demonstrate that the two domains of cTnC tumble independently (average R2 10 s−1), being connected by a flexible linker. However, upon addition of cTnI1-77, the complex tumbles as a rigid unit (R2 30 s−1). cTnI phosphomimetic mutants S22D/S23D, S41D/S43D and dilated cardiomyopathy- (DCM-)associated mutations cTnI K35Q, cTnC D75Y, and cTnC G159D destabilize the active orientation of cNTnC, with intermediate 15N R2 rates (R2 17–23 s−1). The active orientation of cNTnC is stabilized by the flexible tails of cTnI, cTnI1-37 and cTnI135-209. Surprisingly, when cTnC is incorporated into complexes lacking these tails (cTnC-cTnI38-134, cTnC-cTnT223-288, or cTnC-cTnI38-134-cTnT223-288), the cNTnC domain is still immobilized, revealing a new interaction between cNTnC and the IT-arm that stabilizes a “dormant” orientation. We propose that the calcium sensitivity of the cardiac troponin complex is regulated by an equilibrium between active and dormant orientations, which can be shifted through post-translational modifications or DCM-associated mutations.

Patients with coronary heart disease, dilated cardiomyopathy and idiopathic ventricular tachycardia share overlapping patterns of pathogenic variation in cardiac risk genes

BackgroundVentricular tachycardia (VT) is a major cause of sudden cardiac death (SCD). Clinical investigations can sometimes fail to identify the underlying cause of VT and the event is classified as idiopathic (iVT). VT contributes significantly to the morbidity and mortality in patients with coronary artery disease (CAD) and dilated cardiomyopathy (DCM). Since mutations in arrhythmia-associated genes frequently determine arrhythmia susceptibility screening for disease-predisposing variants could improve VT diagnostics and prevent SCD in patients.MethodsNinety-two patients diagnosed with coronary heart disease (CHD), DCM, or iVT were included in our study. We evaluated genetic profiles and variants in known cardiac risk genes by targeted next generation sequencing (NGS) using a newly designed custom panel of 96 genes. We hypothesized that shared morphological and phenotypical features among these subgroups may have an overlapping molecular base. To our knowledge, this was the first study of the deep sequencing of 96 targeted cardiac genes in Kazakhstan. The clinical significance of the sequence variants was interpreted according to the guidelines developed by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) in 2015. The ClinVar and Varsome databases were used to determine the variant classifications.ResultsTargeted sequencing and stepwise filtering of the annotated variants identified a total of 307 unique variants in 74 genes, totally 456 variants in the overall study group. We found 168 mutations listed in the Human Genome Mutation Database (HGMD) and another 256 rare/unique variants with elevated pathogenic potential. There was a predominance of high- to intermediate pathogenicity variants in LAMA2, MYBPC3, MYH6, KCNQ1, GAA, and DSG2 in CHD VT patients. Similar frequencies were observed in DCM VT, and iVT patients, pointing to a common molecular disease association. TTN, GAA, LAMA2, and MYBPC3 contained the most variants in the three subgroups which confirm the impact of these genes in the complex pathogenesis of cardiomyopathies and VT. The classification of 307 variants according to ACMG guidelines showed that nine (2.9%) variants could be classified as pathogenic, nine (2.9%) were likely pathogenic, 98 (31.9%) were of uncertain significance, 73 (23.8%) were likely benign, and 118 (38.4%) were benign. CHD VT patients carry rare genetic variants with increased pathogenic potential at a comparable frequency to DCM VT and iVT patients in genes related to sarcomere function, nuclear function, ion flux, and metabolism.ConclusionsIn this study we showed that in patients with VT secondary to coronary artery disease, DCM, or idiopathic etiology multiple rare mutations and clinically significant sequence variants in classic cardiac risk genes associated with cardiac channelopathies and cardiomyopathies were found in a similar pattern and at a comparable frequency.

Identification of Target Genes and Transcription Factors in Mice with LMNA-Related Dilated Cardiomyopathy by Integrated Bioinformatic Analyses.

BackgroundDilated cardiomyopathy (DCM), which is characterized by enlarged ventricular dimensions and systolic dysfunction, is the most common type of cardiomyopathy. Mutations in the LMNA gene are reported in approximately 10% of familial DCM cases. However, the mechanism of LMNA mutations in human DCM remains unclear.Material/MethodsWe used the GSE36502 and GSE123916 datasets to obtain gene expression profiles from LMNA-related DCM mice and to identify differentially expressed genes (DEGs). Crucial function and pathway enrichment analyses of DEGs were performed. Protein–protein interaction (PPI) network analysis was carried out to identify the top 10 hub genes, which were validated using reverse transcription-polymerase chain reaction (RT-PCR) to find target genes. Weighted gene co-expression network analysis (WGCNA) was used to explore the module relevant to external traits of LMNA-related DCM mice. Transcription factors (TFs) for the selected genes were analyzed using NetworkAnalyst.ResultsA total of 156 common DEGs (co-DEGs) were identified, including 80 up-regulated and 76 down-regulated genes. The enriched biological functions and pathways were oxidative stress, regulation of apoptosis, regulation of fibrosis, and MAPK pathways. Five target genes (Timp1, Hmox1, Spp1, Atf3, and Adipoq) were verified after RT-PCR. Most co-DEGs were discovered to be related to the development of external traits. Three TFs (ELF1, ETS1, and NRF1) showed close interactions with the hub genes.ConclusionsOur study used integrated bioinformatic analyses and revealed some important genes in mice with LMNA-related DCM, which could provide novel insights into the mechanism underlying human LMNA-related DCM.

Troponin destabilization impairs sarcomere-cytoskeleton interactions in iPSC-derived cardiomyocytes from dilated cardiomyopathy patients

The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation. We previously reported that induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with a dilated cardiomyopathy (DCM) mutation, troponin T (TnT)-R173W, display sarcomere protein misalignment and impaired contractility. Yet it is not known how TnT mutation causes dysfunction of sarcomere microdomains and how these events contribute to misalignment of sarcomeric proteins in presence of DCM TnT-R173W. Using a human iPSC-CM model combined with CRISPR/Cas9-engineered isogenic controls, we uncovered that TnT-R173W destabilizes molecular interactions of troponin with tropomyosin, and limits binding of PKA to local sarcomere microdomains. This attenuates troponin phosphorylation and dysregulates local sarcomeric microdomains in DCM iPSC-CMs. Disrupted microdomain signaling impairs MYH7-mediated, AMPK-dependent sarcomere-cytoskeleton filament interactions and plasma membrane attachment. Small molecule-based activation of AMPK can restore TnT microdomain interactions, and partially recovers sarcomere protein misalignment as well as impaired contractility in DCM TnT-R173W iPSC-CMs. Our findings suggest a novel therapeutic direction targeting sarcomere- cytoskeleton interactions to induce sarcomere re-organization and contractile recovery in DCM.

Dilated cardiomyopathy mutation in the converter domain of human cardiac myosin alters motor activity and response to omecamtiv mecarbil

We investigated a dilated cardiomyopathy (DCM) mutation (F764L) in human β-cardiac myosin by determining its motor properties in the presence and absence of the heart failure drug omecamtive mecarbil (OM). The mutation is located in the converter domain, a key region of communication between the catalytic motor and lever arm in myosins, and is nearby but not directly in the OM-binding site. We expressed and purified human β-cardiac myosin subfragment 1 (M2β-S1) containing the F764L mutation, and compared it to WT with in vitro motility as well as steady-state and transient kinetics measurements. In the absence of OM we demonstrate that the F764L mutation does not significantly change maximum actin-activated ATPase activity but slows actin sliding velocity (15%) and the actomyosin ADP release rate constant (25%). The transient kinetic analysis without OM demonstrates that F764L has a similar duty ratio as WT in unloaded conditions. OM is known to enhance force generation in cardiac muscle while it inhibits the myosin power stroke and enhances actin-attachment duration. We found that OM has a reduced impact on F764L ATPase and sliding velocity compared with WT. Specifically, the EC50 for OM induced inhibition of in vitro motility was 3-fold weaker in F764L. Also, OM reduces maximum actin-activated ATPase 2-fold in F764L, compared with 4-fold with WT. Overall, our results suggest that F764L attenuates the impact of OM on actin-attachment duration and/or the power stroke. Our work highlights the importance of mutation-specific considerations when pursuing small molecule therapies for cardiomyopathies.

Disrupted mechanobiology links the molecular and cellular phenotypes in familial dilated cardiomyopathy

Familial dilated cardiomyopathy (DCM) is a leading cause of sudden cardiac death and a major indicator for heart transplant. The disease is frequently caused by mutations of sarcomeric proteins; however, it is not well understood how these molecular mutations lead to alterations in cellular organization and contractility. To address this critical gap in our knowledge, we studied the molecular and cellular consequences of a DCM mutation in troponin-T, ΔK210. We determined the molecular mechanism of ΔK210 and used computational modeling to predict that the mutation should reduce the force per sarcomere. In mutant cardiomyocytes, we found that ΔK210 not only reduces contractility but also causes cellular hypertrophy and impairs cardiomyocytes' ability to adapt to changes in substrate stiffness (e.g., heart tissue fibrosis that occurs with aging and disease). These results help link the molecular and cellular phenotypes and implicate alterations in mechanosensing as an important factor in the development of DCM.

Abstract 908: Mechanism of Calcium Sensitivity Modulation of Cardiac Troponin C by Small Molecules Illuminated by Umbrella Sampling Simulations

Cardiac troponin C (cTnC) binds intracellular calcium and subsequently cardiac troponin I (cTnI), initiating cardiac muscle contraction. Due to its role in contraction, cTnC has been a therapeutic target in the search for small molecules to treat cardiomyopathies that interfere with normal muscle contraction. Structural studies have shown the binding location of small molecules such as bepridil, dfbp-o, 3-methyldiphenylamine (DPA) and W7 to be a hydrophobic pocket in the regulatory domain of cTnC (cNTnC) but have not shown the influence of these small molecules on the dynamics of opening this domain. Here we describe an application of an umbrella sampling method used to elucidate the impact these calcium sensitivity modulators have on the free energy of cNTnC hydrophobic patch opening. We found that all these molecules lowered the free energy of opening in the absence of the cTnI, with bepridil facilitating the least endergonic transformation. In the presence of cTnI, however, we saw a stabilization of the open configuration due to DPA and dfbp-o binding, and a destabilization of the open configuration imparted by bepridil and W7. Predicted poor binding molecule NSC34337 left the hydrophobic immediately in conventional MD simulations suggesting that only hydrophobic patch binders stabilized the open conformation. Additionally, differences in the free energy of hydrophobic patch opening of hypertrophic (HCM) and dilated cardiomyopathy (DCM) cTnC systems were investigated. Molecular dynamics and umbrella sampling simulations revealed a lower free energy of opening for the HCM mutations A8V and A31S, as well as the calcium-sensitizing mutation L48Q. The DCM mutations, Y5H, Q50R, and E59D/D75Y, all exhibited a higher free energy of opening. In conclusion, our developed simulation protocol presents a novel approach to study calcium sensitivity modulation by small molecules and mutations and facilitates a molecular understanding of cardiac muscle contraction.